Overview: This research program addresses basic molecular and physiological processes of nociceptive (pain-sensing) transmission in the peripheral and central nervous systems and new ways to effectively control pain. The molecular research is performed using animal and in vitro cell-based models. We concentrate on primary afferent pain-sensing neurons located in dorsal root ganglion (DRG) that send nerve fibers to skin and deep tissues and make connections within dorsal spinal cord, which is the first site of synaptic information processing for pain. The mechanisms of transduction of physical pain stimuli in DRG fibers are investigated through models of pathophysiological damage or using reductionist preparations such as primary DRG cultures or heterologous expression systems of ion channels or receptors. Our goals are (a) to understand the molecular and cell biological mechanisms of acute and chronic pain at the initial steps in the pain pathway, (b) to investigate mechanisms underlying human chronic pain disorders, (c) to explore neuronal plasticity and altered gene expression in persistent pain states, and (d) to use this knowledge to devise new treatments for pain. New Treatments for Pain: We address the new treatment goal by a translational research and human clinical trials program aimed at developing new molecular interventions for severe pain. Studies with the TRPV1 agonist resiniferatoxin (RTX) have resulted in a Phase I clinical trial of RTX for pain treatment in patients with advanced cancer. RTX activates TRPV1, which is an inflammation- and heat-sensitive calcium/sodium ion channel that normally converts painful heat or acidic pH into nerve action potentials by opening the pore of the ion channel. The influx of ions depolarizes pain-sensing nerve endings and sends electrical signals to the spinal cord (which in turn sends the signals to the brain where we perceive pain). RTX is a potent capsaicin analog that props open the TRPV1 ion channel, causing calcium cytotoxicity. Depending on the route of administration this disables or deletes TRPV1 pain-sensing neurons, their nerve endings or axons (i.e., the nerve fiber). RTX produces very effective pain control in pre-clinical models. The central route involves administration into the cerebrospinal fluid around the spinal cord (intrathecal). We can also inject directly into sensory ganglia, both routes produce permanent effects because TRPV1-containing neurons or axons are killed. For cancer pain, after IRB approval of the clinical protocol and the Investigational New Drug application by the FDA, we treated several patients with severe pain from advanced cancer. Each patient is a unique case since the tumor presents and progresses differently in each one. Thus, multiple endpoints for determining efficacy are necessary. Peripheral routes of RTX administration include direct injection into skin, joints, nerve bundles, or topically onto the cornea. Analgesia by these routes can be long-lasting but temporary since nerve endings and axons regrow. We are also in the process of generating new protocols for post-herpetic neuralgia, localized cancer pain, and osteoarthritis (OA). The OA project is incentivized by observations of strong efficacy and long duration of action (4 to >12 months) upon RTX injection into knee joints of clien-owned dogs with OA. These results reinforce the objective of therapeutic use of vanilloid agonists for pain control and the translation to human patients. Early Translational Investigations: A new approach to using TRPV1 as an analgesic target involves identification of small chemicals that act as positive allosteric modulators (PAMs) of TRPV1 activation. Our lab developed a high throughput calcium fluorescence assay for TRPV1 PAMs and we screened the Molecular Libraries small molecule collection at the NIH Chemical Genomics Center of NCATS. We discovered several new chemical compounds that enhance the activation of TRPV1 upon orthosteric agonist (capsaicin) binding or by elevated H+ ions, consistent with positive modulation of the TRPV1 ion channel open state. Further medicinal chemical modifications of one lead compound identified an analog, DPM-32. This was tested in vivo for nerve terminal inactivation and was active (results published). These results represent the first in vivo observations of TRPV1 positive allosteric modulation. Further modification to increase metabolic stability yielded DPM-74. This was shown to enhance neuropeptide release from rat primary DRG cultures. In collaboration, we have also examined TRPV1 agonist activity of various polyunsaturated ethanolamines and fatty acids, which comprise one of the major classes of putative endovanilloids. These could function as the endogenous orthosteric agonist at TRPV1. The present studies reveal the existence of a new class of pharmacological agents for pain modulation and pain control. Our investigations support the idea that TRPV1 agonist activity, induced in several different ways, has the potential to yield novel, non-narcotic, non-addictive, selective, long-lasting analgesic agents that may be effective in acute, persistent, or chronic pain disorders. Basic Pain Mechanisms: Underlying the translational and clinical studies are investigations of molecular biology, neuronal function, behavior, and mechanisms of pain transduction. We are systematically investigating molecular alterations at the first three steps in the pain pathway beginning with injured peripheral tissue, the dorsal root ganglion and the dorsal (sensory) spinal cord. One goal of this approach is to obtain a comprehensive, quantitative molecular understanding of nociceptive processes related to inflammation and nerve injury. Our studies reveal a complex, dynamic modulation of gene expression and tissue cellular composition at all three steps. We have identified prominent roles for new, key molecules with distinct combinatorial patterns of expression among the three tissues. We are using a method called RNA-seq to sequence all of the mRNAs in a given tissue or cell population. This method is sensitive and quantitative. We also combine it with genetically or pharmacologically manipulated mice and rats to obtain specific cell population for sequencing. The results have proven to be both compelling and informative, and allow us to define the pain transducing genetic complement of specific sensory ganglion neuronal populations. We also have used it to define genes involved in inherited peripheral neuropathies and pain channelopathies. We now have a much better quantitative assessment of genes expressed in the multiple tissues of the nociceptive circuit, in the periphery and under pathophysiological conditions. Through this basic research we shall obtain a deeper understanding of mechanisms that trigger and sustain chronic pain.